Breast cancers that are triple negative for estrogen receptor, progesterone receptor, and Her2 (thereafter referred as TNBC) account for 15-20% of breast carcinomas and are notoriously aggressive, yet there is no targeted therapy against these malignancies. Basal-like (BLBC) and claudin-low (CLBC) are the two major subtypes of TNBC. CLBCs show characteristic of epithelial-to-mesenchymal transition (EMT) and are thought to originate from mammary stem cells (MaSC). Notch is a powerful regulator for stem cells as well as EMT in many tissues including the mammary gland. Most recently we have revealed that Lunatic Fringe (Lfng), one of the mammalian Fringes that modify Notch receptors and modulate Notch activation, exerts tumor suppressor function in MaSC and luminal progenitor cells. Interestingly, while Lfng expression is lowest in BLBC among various subtypes of human breast cancer, another mammalian Fringe, Manic Fringe (Mfng), shows highest expression in CLBC. Thus Fringes may differentially regulate Notch activation in different cell types of mammary epithelial hierarchy, and genetic alterations of Fringes may cause dysregulation of Notch signaling in different cells of origin, ultimately leading to different subtypes of breast cancer. While Lfng suppresses BLBC, Mfng may facilitate CLBC initiation and/or progression. Indeed, our preliminary analysis in a CLBC cell line suggests that Mfng enhances Notch4 signaling, which has been shown to regulate breast cancer stem cell activity. In this proposal, we plan to investigate Mfng roles in CLBC with the following Specific Aims: 1) Define Mfng functions using breast cancer cell lines. We will knockdown Mfng in CLBC cell lines and overexpress Mfng in non-CLBC cell lines to test for effects on Notch activation as well as EMT, cancer stem cell enrichment and its tumor initiating and metastatic capacity. 2) Identify potential downstream targets of Mfng in CLBC. We intend to perform microarray gene expression analysis in stable cell lines with Mfng knockdown or overexpression (from Aim 1), and to examine the expression of candidate genes in CLBC tissues. 3) Validate Mfng roles in CLBC mouse models. We will breed the Mfng null mice into CLBC mouse models to determine whether loss of Mfng suppresses CLBC tumor formation. We hope to obtain data to demonstrate that Mfng promotes CLBC initiation and/or progression. As a druggable enzyme, Mfng could be an ideal CLBC target since it affects specific cell types rather than broad impact from overall inhibition of Notch signaling. Furthermore, understanding the unique biology of CLBC and specific roles of Mfng-modulated Notch activation in CLBC will shed insight on cancer stem cell, which is thought to initiate recurrence and metastasis.